Significant research efforts are ongoing to elucidate the complex molecular mechanisms underlying amyotrophic lateral sclerosis (ALS), which may in turn pinpoint potential therapeutic targets for treatment. The ALS research field has evolved with recent discoveries of numerous genetic mutations in ALS patients, many of which are in genes encoding RNA binding proteins (RBPs), including TDP-43, FUS, ATXN2, TAF15, EWSR1, hnRNPA1, hnRNPA2/B1, MATR3 and TIA1. Accumulating evidence from studies on these ALS-linked RBPs suggests that dysregulation of RNA metabolism, cytoplasmic mislocalization of RBPs, dysfunction in stress granule dynamics of RBPs and increased propensity of mutant RBPs to aggregate may lead to ALS pathogenesis. Here, we review current knowledge of the biological function of these RBPs and the contributions of ALS-linked mutations to disease pathogenesis.

Keywords: ALS, RNA-binding proteins

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord, leading to muscle weakness, paralysis and death (Rowland and Shneider, 2001). Although the underlying cause is unknown for most ALS cases, advances in sequencing technology have allowed the identification of more than one hundred genes associated with ALS, including approximately thirty potential ALS-causing drivers (Al-Chalabi et al., 2017; Guerreiro et al., 2015; Wroe et al., 2008). Interestingly, many of the genes implicated in ALS encode RNA-binding proteins (RBPs), which include transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS), ataxin-2 (ATXN2), TATA-box binding protein associated factor 15 (TAF15), Ewing’s sarcoma breakpoint region 1 (EWSR1), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), matrin 3 (MATR3) and T-cell-restricted intracellular antigen-1 (TIA1). As shown in Fig. 1, these RBPs share structural similarities; they all contain one or more RNA-binding domains (e.g., RRM, Lsm or LsmAD), a glycine (Gly)-rich region (except for MATR3 and ATXN2), and a nuclear localization signal (NLS) (except for TIA1 and ATXN2). In addition, these RBPs share functional similarities, since they are involved in RNA metabolism and many localize to stress granules upon cellular stress. Stress granules are membrane-less discrete cytoplasmic structures containing mRNA and associated proteins that form as a protective response to stress (Buchan and Parker, 2009; Kedersha et al., 1999; Monahan et al., 2016). Interestingly, the stress granule proteome was shown to be enriched for proteins encoding predicted prion-like or low-complexity (LC) domains, including many ALS-linked RBPs (Jain et al., 2016; Monahan et al., 2016). Mutations within these LC domains have been shown to interrupt stress granule dynamics and increase aggregation or fibrillization, which suggests a potential mechanism for ALS pathogenesis. Indeed, an increasing number of mutations identified in RBPs strongly suggest that abnormal RBP function and dysregulated RNA homeostasis also lead to ALS pathogenesis. Much progress has been made in understanding the mechanisms of how mutant RBPs exert toxicity and lead to neurodegeneration in ALS. In this review, we summarize studies that have investigated the biological function of the potential ALS-driving RBPs and the mechanisms by which mutations in these RBPs cause ALS. Where available, we discuss what we have learned from ALS models, with a focus on mouse models.

In 2006, TDP-43 was identified as a major component of protein inclusions in the cytoplasm of ALS-affected motor neurons, marking it as the first RBP associated with ALS (Arai et al., 2006; Mackenzie et al., 2007; Neumann et al., 2006). Corresponding with abnormal cytoplasmic inclusion, nuclear clearance of TDP-43 was also observed (Neumann et al., 2006; Van Deerlin et al., 2008). This finding raises the question of whether TDP-43 pathogenicity is due to loss of nuclear function, gain of cytoplasmic function, or both. Numerous TDP-43 mutations have been identified in ALS patients, accounting for approximately 5% of familial and less than 1% of sporadic cases (Taylor et al., 2016). Most mutations are clustered in the Gly-rich domain (Fig. 1) (Kapeli et al., 2017; Sreedharan et al., 2008). The Gly-rich, LC domain has been shown to be necessary for TDP-43 assembly into stress granules (Colombrita et al., 2009) and for undergoing liquid-liquid phase separation (or phase separation) (Aguilera-Gomez and Rabouille, 2017; Anderson and Kedersha, 2009; Brangwynne et al., 2009; Chong and Forman-Kay, 2016; Conicella et al., 2016). This process is dynamic, as proteins can transition between liquid and gel-like states (recently reviewed in (Purice and Taylor, 2018)). However, TDP-43 proteins that harbor disease mutations in the LC domain were shown to favor the gel-like, rigid state and form aggregates (Conicella et al., 2016). Additionally, these mutant TDP-43 proteins increased the formation of stress granules upon oxidative stress induced by sodium arsenite (Liu-Yesucevitz et al., 2010). As many other studies have demonstrated that ALS mutations in the LC domain results in increased conversion to aggregates (Kapeli et al., 2017; Purice and Taylor, 2018), phase separation disruption is a possible mechanism leading to ALS. In addition to full length TDP-43, low molecular weight species containing the LC domain (e.g., TDP-35) were also found to localize to cytoplasmic inclusions in ALS motor neurons (Xiao et al., 2015), further supporting that this LC domain is critical for aggregation. In parallel, other studies have shown that dysregulation in RNA splicing due to loss of TDP-43 function may be involved in ALS pathogenesis. One example demonstrated by Ling and colleagues showed that cryptic exon inclusion induced by loss of TDP-43 is found in ALS patients presenting TDP-43 pathology (Ling et al., 2015).

Many transgenic mice and rats expressing mutant TDP-43 have been generated; most of which reproduce clinical features seen in ALS patients including impaired motor function, degeneration of motor neurons, and accumulation of ubiquitinated TDP-43 cytoplasmic aggregates (Liu et al., 2013; Picher-Martel et al., 2016; Wegorzewska et al., 2009). Loss of TDP-43 was found to cause age-dependent motor neuron degeneration in mice and many other animal models (Iguchi et al., 2013; Vanden Broeck et al., 2014), suggesting that loss of TDP-43 function is involved in ALS pathogenesis. However, it is still not clear to what magnitude gain-of-function and loss-of-function contribute to neurodegeneration.

Mutations in FUS were reported to cause ALS in 2009 (Kwiatkowski et al., 2009; Vance et al., 2009). FUS is normally localized primarily in the nucleus, but in ALS-affected brains and spinal cords, FUS is often found aggregated in the cytoplasm. Interestingly, TDP-43 pathology is absent in ALS patients with FUS mutations (Vance et al., 2009). FUS mutations account for 5% of familial ALS and less than 1% of sporadic ALS cases (Taylor et al., 2016). The average age of onset for ALS cases with FUS mutations is 43.6 ± 15.8 years, which is relatively earlier than that of patients with SOD1 or TDP-43 mutations (Shang and Huang, 2016; Yan et al., 2010). In addition, many FUS mutations were found in juvenile ALS cases (late teens and early 20s) (Baumer et al., 2010; Huang et al., 2010; Liu et al., 2017b). Most of the mutations identified in FUS are clustered in either the N-terminal glutamine-glycine-serine-tyrosine (QGSY)-rich and Gly-rich regions or the C-terminal region within the arginine-glycine-glycine (RGG)-rich domain and NLS (Fig. 1). Several groups have demonstrated that ALS mutations accelerate the phase transition of FUS and readily lead to formation of nuclear and cytoplasmic aggregates (Murakami et al., 2015; Patel et al., 2015). In addition, ALS-linked FUS mutants have shown increased localization to stress granules upon cellular stress (Bosco et al., 2010). Recently, the detailed structural and molecular mechanism of the phase behavior of FUS has been further investigated (Hofweber et al., 2018; Luo et al., 2018; Murray et al., 2017; Qamar et al., 2018; Yoshizawa et al., 2018), providing additional support for aberrant phase transition as a molecular mechanism leading to disease.

Two mutations within the RGG-rich domain of EWSR1 were identified in sporadic ALS cases in 2012 (Couthouis et al., 2012). The ALS-associated mutations were found to promote cytoplasmic accumulation of mutant EWSR1 in primary mouse neuron cultures and increase aggregation kinetics when compared to the wild-type protein. In Drosophila, overexpression of wild-type EWSR1 leads to neurodegeneration; however, overexpression of mutant EWSR1 does not exacerbate the phenotypes. In postmortem tissue from sporadic ALS patients, EWSR1 was present in cytoplasmic punctate granular structures (Couthouis et al., 2012). Together, these results suggest that EWSR1 has a potential role in conferring toxicity, but further experiments on these mutations and additional studies to identify novel mutations in EWSR1 are required.

Mutations in hnRNPA1 underlie less than 1% of ALS cases (Taylor et al., 2016). Kim and colleagues identified hnRNPA1 missense mutations in a family affected by ALS and in two families with multisystem proteinopathy (MSP) affecting the brain, motor neurons, muscle and bone (Kim et al., 2013). Cytoplasmic accumulation and nuclear clearance of mutant hnRNPA1 were observed in patient muscle tissue (Kim et al., 2013). Interestingly, hnRNPA1 staining in the postmortem tissue of sporadic ALS patients was reduced in the nuclei of motor neurons relative to that of control tissue, and did not colocalize with TDP-43 inclusions (Honda et al., 2015). ALS-associated mutations in the Gly-rich LC domain of hnRNPA1, which mediates phase separation (Molliex et al., 2015), have been shown to increase incorporation into stress granules, strengthen steric zipper motifs and accelerate fibrillization compared to wildtype hnRNPA1 (Kim et al., 2013; Molliex et al., 2015). Additional mutations in hnRNPA1 were later identified by targeted sequencing of sporadic ALS patients and of an ALS family with flail arm syndrome (Couthouis et al., 2014; Liu et al., 2016). HnRNPA1 knockout mice are embryonic lethal, while heterozygous animals display a cardiac phenotype and show many changes in alternative splicing of muscle development-related genes (Liu et al., 2017a). This evidence supports an important role for hnRNPA1 in alternative splicing, but further animal studies are required to investigate its role in ALS pathogenesis.

A MATR3 mutation (S85C) was first identified in autosomal-dominant distal myopathy (Senderek et al., 2009). However, in 2014, myopathy in patients with the S85C mutation was reclassified as ALS, and other missense mutations in MATR3 were identified in familial ALS cases (Johnson et al., 2014). Since then, several other missense and splicing mutations have been identified in familial and sporadic ALS cases, although mutations in MATR3 account for less than 1% of all ALS cases (Leblond et al., 2016; Lin et al., 2015; Marangi et al., 2017; Origone et al., 2015; Taylor et al., 2016; Xu et al., 2016). None of these mutations are found in known domains; instead, they are clustered in two regions as shown in Fig. 1. MATR3 staining in ALS postmortem spinal cord tissue is primarily observed in the nucleus. However, in some cases, diffuse cytoplasmic MATR3 or MATR3 inclusions have been observed (Johnson et al., 2014; Tada et al., 2018). For example, intense nuclear and diffuse cytoplasmic MATR3 staining was observed in a patient harboring a MATR3 F115C mutation (Johnson et al., 2014). A very recent study found MATR3 in a subset of cytoplasmic TDP-43-positive inclusions (Tada et al., 2018). However, several studies demonstrated that wild-type or mutant MATR3 was mostly localized in the nucleus even upon stress and that only a small subset of cells overexpressing MATR3 show cytoplasmic puncta resembling stress granules (Gallego-Iradi et al., 2015). A recent paper showed that fibroblasts from S85C myopathy patients exhibited no significant changes in mutant MATR3 localization but showed impaired stress granule formation in response to stress, suggesting that mutant MATR3 may indirectly impact stress granule formation (Mensch et al., 2018). Several recent studies have attempted to identify protein interactors of both wild-type and mutant MATR3; one study found an enrichment of proteins involved in mRNA nuclear export to interact with wild-type MATR3, and these proteins show altered interactions with the mutant form (Boehringer et al., 2017). In contrast, another study did not find significant differences in the interactions with binding partners between wild-type and mutant MATR3 (Iradi et al., 2018). Further studies are required to determine how MATR3 mutations confer pathogenicity.

Several RBPs have been identified to be strongly linked with ALS. Many of these proteins share structural and functional properties that mediate their role in the disease process. The most striking structural property shared by many of the RBPs are the LC domains. When harboring ALS-linked mutations in these domains, these RBPs are associated with increased aggregation or fibrillization propensity, cytoplasmic mislocalization, and dysregulation of stress granule dynamics, suggesting that LC domains play an important role in ALS pathogenesis. However, not all ALS-linked RBPs have a defined LC domain (e.g., MATR3 and ATXN2), suggesting that alternative pathogenic mechanisms may exist. By definition, these RBPs play functional roles in RNA metabolism, including transcription, RNA processing, mRNA export and stability, and translation regulation. As such, ALS-linked mutations in these proteins have the potential to affect gene expression and thereby impact certain cellular processes, including the DNA repair response, apoptosis, and cell growth and proliferation. However, it is difficult to pinpoint a single or a few pathway(s) or mechanism(s) by which all these RBPs converge to cause ALS. A better understanding of the normal function as well as pathological significance of these RBPs will be critical to illuminate the biology behind this devastating disease.